Quantification of α-Gal Antigen Removal in the Porcine Dermal Tissue by α-Galactosidase

• Published in: Tissue engineering. Part C, Methods Methods, 2015-11, Vol.21 (11), p.1197-1204
• Main Authors: Gao, Hong-Wei, Li, Su-Bo, Sun, Wendell Q., Yun, Zhi-Min, Zhang, Xue, Song, Jin-Wen, Zhang, Shi-Kun, Leng, Ling, Ji, Shou-Ping, Tan, Ying-Xia, Gong, Feng
• Format: Article
• Language: English
• Subjects: alpha-Galactosidase - metabolism; Animals; Antibodies, Monoclonal - metabolism; Antigens - metabolism; Cattle; Dermis - metabolism; Enzyme-Linked Immunosorbent Assay; Epitopes - immunology; Fluorescent Antibody Technique; Galactose - metabolism; Sus scrofa
• ISSN: 1937-3384; 1937-3392
• DOI: 10.1089/ten.tec.2015.0129

The α-Gal (Galα1,3-Galβ1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-man tissue and organ xenotransplantation. The elimination or reduction of the α-Gal epitopes is therefore an important step for a successful xenotransplantation. The present study is to evaluate the α-Gal elimination in the porcine skin with α-galactosidase treatment, and to assess two methods (immunohistochemistry and inhibition ELISA) that may be used in quality control for quantifying the extent of the α-Gal elimination. Enzymatic cleavage in a single-step process is extremely efficient and affordable at eliminating the α-Gal epitope even in a tissue as dense as the porcine dermis. The cost of enzymatic cleavage is found to be less than US$7 for a 10 × 10 cm piece of porcine skin (0.5 mm thick) or about US$140 for 100 g of 3-dimensional soft tissues. After enzymatic cleavage, the α-Gal-positive immunostaining was essentially undetectable in enzyme-treated porcine skin. The inhibition rate constant of the monoclonal anti-Gal antibody M86 binding to α-Gal-bovine serum albumin in ELISA was reduced from 15.0 ± 4.3 ( n  = 10) to 6.1 ± 2.6 ( n  = 7) after enzyme treatment, in comparison to 4.4 ± 1.8 ( n  = 9) background inhibition of decellularized human skin (the ultimate negative control), which demonstrates ∼84% elimination of α-Gal epitopes in treated porcine skin. To examine the suitability of two detection methods for the routine quality control application, comparative studies were made with control and enzyme-treated porcine skin, porcine skin from the α-Gal knockout animal, as well as decellularized human skin. The data show that the traditional immunohistochemistry and, to a less extent, the inhibition ELISA with further modifications can be used as quality control tools in the production and selection of biocompatible bioprosthetic devices. The biological evaluation of enzyme-treated porcine skin is ongoing with a small animal model and a nonhuman primate model.